Malaria remains the most prevalent vector-borne disease of man, infecting an estimated 488 million persons yearly. Nearly 15 million of these cases occur in neotropical America transmitted by Anopheles from five species complexes. The biology of many of these species is poorly known, and it is very difficult to separate certain vectors from non-vectors by classical means. This proposal seeks to apply an interdisciplinary approach to determine the genetic affinities and evolutionary relationships in the Anopheles nuneztovari sister group which includes known or suspected vectors of human malaria in Venezuela, Peru, Ecuador, Colombia, Suriname, Brazil and Bolivia. The project will also produce new tools for biosystematic determinations and investigate the importance of multiple blood feeding among related Anopheles. Larval and adult mosquitoes will be collected in the field in six S. American countries and brought live to laboratories in Florida for further study. Link-reared progeny of single females will be examined in all life stages and compared between populations. Scanning electron microscopy will be used to examine mosquito eggs. Intrinsic barriers to hybridization will be tested by induced mating of sympatric and allopatric populations. Larval salivary polytene chromosomes will be studied for inversions and markers which characterize species and populations. Twenty or more isoenzymes will be examined by starch gel electrophoresis for loci that are diagnostic for species or populations. DNA from A. nuneztovari 'sisters' will be extracted, purified, cloned and sequenced. Species and populations will be separated by restriction endonuclease profiles of mtDNA and rDNA and squash blot probes. Phylogenies will be constructed and compared from the morphological, hybridization, chromosomal, isoenzyme, and DNA results. The relationship between blood meal size and the frequency of feeding per gonotrophic cycle will be studied in the laboratory for all populations. A histological technique which identifies multiple blood meals in the mosquito midgut will be used to assess the incidence of multiple feeding in nature and evaluate its significance for malaria transmission.